Early OA Stage Like Response Occurs after Dynamic Stretching of Human Synovial Fibroblasts.
ABSTRACT: As events triggering early osteoarthritis onset can be related to mechanical stress and proinflammatory signaling, we investigated the effect of different mechanical strain protocols on the expression of proinflammatory genes, as well as extracellular matrix remodelling in human synovial fibroblasts. Three distinct models of tensile stretching were applied: static isotropic tensile strain at 0 Hz, 16% tension for 48 h; short-term high-frequency cyclic tension at 1 Hz, 10% tension for 4 h; and dynamic tensile stretching for 48 h, consisting of two blocks of moderate stretching at 0.2 Hz, 2%, advanced stretching at 0.5 Hz, 15%, or a combination of both. General signs of inflammation were present after static isotropic tension, whereas short-term high-frequency cyclic tension showed increased levels of IL-6 paired with diminished levels of IL-1?. Reduced inflammatory effects of TNF-?, IL-6, and IL-1? were observed when exposed to advanced stretching. Long-term tensile strain induced extracellular matrix remodelling at the gene and protein levels. While hyaluronan acid synthesis was increased with static tensile strain, dynamic tensile stretching had a reducing effect. Our study revealed that proinflammatory markers were activated by mechanical strain as seen in static isotropic tension and short-term high-frequency tensile strain, whereas long-term exposure induced extracellular matrix remodelling processes.
Project description:We recently developed a fiber composite consisting of tenocytes seeded onto discontinuous fibers embedded within a hydrogel, designed to mimic physiological tendon micromechanics of tension and shear. This study examined if cell adhesion peptide (DGEA or YRGDS), fiber modulus (50 or 1300?kPa) and/or cyclic strain (5% strain, 1?Hz) influenced bovine tenocyte gene expression. Ten genes were analyzed and none were sensitive to peptide or fiber modulus in the absence of cyclic tensile strain. Genes associated with tendon (SCX and TNMD), collagens (COL1A1, COL3A1, COL11A1), and matrix remodelling (MMP1, MMP2, and TIMP3) were insensitive to cyclic strain. Contrarily, cyclic strain up-regulated IL6 by 30-fold and MMP3 by 10-fold in soft YRGDS fibers. IL6 expression in soft YRGDS fibers was 5.7 and 3.3-fold greater than in soft DGEA fibers and stiff RGD fibers, respectively, under cyclic strain. Our findings suggest that changes in the surrounding matrix can influence catabolic genes in tenocytes when cultured in a complex strain environment mimicking that of tendon, while having minimal effects on tendon and homeostatic genes.
Project description:HDFs were cultured for 24 hrs for adhesion and to reach 50-70% confluence before mechanical tension was applied. Cyclic mechanical stretch (CMS) with a 0.5-Hz sinusoidal curve at 10-20% elongation was applied using a Flexcell○RFX-5000TM Tension System during the stretching. Cells were harvested immediately after the application of CMS stimulation was completed. Control cells were cultured on the same plates in the same incubator with no stretching. Overall design: Examination of ELK1's different bind motifs of HDFs in the control condition and mechanic loading condition.
Project description:Introduction:Tendon tissue engineering requires scaffold-free techniques for safe and long-term clinical applications and to explore alternative cell sources to tenocytes. Therefore, we histologically assessed tendon formation in a scaffold-free Bio-three-dimensional (3D) construct developed from normal human dermal fibroblasts (NHDFs) using our Bio-3D printer system under tensile culture in vitro. Methods:Scaffold-free ring-like tissues were constructed from 120 multicellular spheroids comprising NHDFs using a bio-3D printer. Ring-like tissues were cultured in vitro under static tensile-loading with or without in-house tensile devices (tension-loaded and tension-free groups), with increases in tensile strength applied weekly to the tensile-loaded group. After a 4 or 8-week culture on the device, we evaluated histological findings according to tendon-maturing score and immunohistological findings of the middle portion of the tissues for both groups (n = 4, respectively). Results:Histology of the tension-loaded group revealed longitudinally aligned collagen fibers with increased collagen deposition and spindle-shaped cells with prolonged culture. By contrast, the tension-free group showed no organized cell arrangement or collagen fiber structure. Additionally, the tension-loaded group showed a significantly improved tendon-maturing score as compared with that for the tension-free group at week 8. Moreover, immunohistochemistry revealed tenascin C distribution with a parallel arrangement in the tensile-loading direction at week 8 in the tension-loaded group, which exhibited stronger scleraxis-staining intensity than that observed in the tension-free group at weeks 4 and 8. Conclusions:The NHDF-generated scaffold-free Bio-3D construct underwent remodeling and formed tendon-like structures under tensile culture in vitro.
Project description:During orthodontic tooth movement (OTM) to therapeutically correct the position of misaligned teeth, thus improving oral health and quality of life, fibroblasts, macrophages, and other immune cells within the periodontal ligament (PDL), which connects a tooth to its surrounding bone, are exposed to compressive and tensile strain. While it is known that PDL fibroblasts are critically involved in the biological regulation of OTM by a mechanotransductively triggered release of cytokines, it is unclear whether macrophages also react to pressure and tension in a similar manner thus impacting on or mediating OTM. RAW264.7 macrophages were seeded onto conventional 6-well cell culture plates for pressure or on Bioflex plates for tension assays and preincubated for 24?h. For in vitro simulation of physiological orthodontic compressive or tensile strain for 2?h, 4?h, 24?h, and 48?h, glass discs (2?g/cm2) were placed or adherent macrophages isotropically stretched for 16%, respectively. We determined cell number, cytotoxicity, and gene/protein expression of Vegf-a/VEGF-A (macrophage-mediated angiogenesis), Mmp-8/9 (extracellular matrix reorganization), and Cox-2/PG-E2, Il-6/IL-6, and Tnf-?/TNF-? (proinflammatory mediators) by RT-qPCR and ELISA. Compressive but not tensile strain resulted in a significant reduction in cell number after only 2?h. Mmp-8 and Mmp-9 expression was significantly enhanced within 24?h of compressive and in part tensile strain. Significantly increased Vegf-a/VEGF-A expression was detected within 4?h of pressure, but not during application of tensile strain. Expression of proinflammatory mediators Cox-2/PG-E2, Il-6/IL-6, and Tnf-?/TNF-? was significantly increased as early as 2-4?h after application of compressive or tensile strain. Our results indicate that macrophages respond early on to compressive and tensile strain occurring during OTM with an enhanced gene expression of proinflammatory cytokines, which could affect PDL fibroblasts, osteoblasts, and immune cells triggering or enhancing the biological mechanisms and osteoclastogenesis underlying OTM.
Project description:To investigate effects of excessive mechanical loading on chondrocytes. Overall design: Primary chondrocytes were isolated from 6-day-old C57BL/6J mice, and were subjected to cyclic tensile strain (0.5 Hz, 10% elongation) for 30 min. Total RNA was isolated from the mouse primary chondrocytes before and 24 hours after the cyclic tensile strain loading.
Project description:Understanding how cells respond to and cope with high levels of mechanical stress is important to gain a better understanding of mechano-biology, both in health and disease. The experiment was designed to assess the total mRNA levels in mechanically stimulated cells and was used, in conjunction with mass spectrometry data, to gain an insight into the systemic response to high-intensity mechanical strain. Here, total RNA was extracted from human mesenchymal stem cells (n=6) following 1 hour of cyclic tensile strain (2.6 – 6.2% strain, 5.0 Hz) using a Flexcell Tension Plus device. RNA from unstrained cells was used as control. The protein coding RNAs were then sequenced with Illumina HiSeq technology.
Project description:INTRODUCTION:Mechanical forces play critical roles in the development and remodelling process of bone. As an alternative cell source for bone engineering, adipose-derived stem cells (ASCs) should be fully investigated for their responses to mechanical stress and the mechanisms responsible for osteogenic induction in response to mechanical signals. MATERIAL AND METHODS:We hypothesized that appropriate application of uniaxial cyclic tensile strain to ASCs could increase bone morphogenetic protein-2 (BMP-2) expression and improve osteogenesis of ASCs. To test our hypothesis, ASCs from the same flask of the same donor were subjected to tensile strain with different patterns in order to eliminate the difference of donor site and passage. After surface markers investigation, the osteo-induced ASCs were subjected to uniaxial cyclic tensile stretch with the following two loading patterns: long duration continuous pattern (6 h, 1 HZ, 2000 µ?) and short duration consecutive pattern (17 min every day for 10 consecutive days, 1 HZ, 2000 µ?). Then osteogenic related genes were analysed by real-time PCR. RESULTS:The ASCs were positive for the markers STRO-1, CD90 and CD44 and negative for CD34. Cyclic tensile strain of 6 continuous h' duration significantly increased gene expressions of BMP-2 and Runx2, and depressed OCN mRNA expression. In contrast, mechanical loading of 17 min every day did not significantly affect gene expression of BMP-2, Runx2, OCN or ALP. CONCLUSIONS:We indicate that ASCs may sense mechanical loading in a duration-dependent manner and cyclic tensile stretch may modulate the osteogenic differentiation of ASCs via the BMP-2 signalling pathway.
Project description:Scaffold-free systems have emerged as viable approaches for engineering load-bearing tissues. However, the tensile properties of engineered tissues have remained far below the values for native tissue. Here, by using self-assembled articular cartilage as a model to examine the effects of intermittent and continuous tension stimulation on tissue formation, we show that the application of tension alone, or in combination with matrix remodelling and synthesis agents, leads to neocartilage with tensile properties approaching those of native tissue. Implantation of tension-stimulated tissues results in neotissues that are morphologically reminiscent of native cartilage. We also show that tension stimulation can be translated to a human cell source to generate anisotropic human neocartilage with enhanced tensile properties. Tension stimulation, which results in nearly sixfold improvements in tensile properties over unstimulated controls, may allow the engineering of mechanically robust biological replacements of native tissue.
Project description:Theoretical works suggest the possibility and usefulness of strain engineering of graphene by predicting remarkable properties, such as Dirac cone merging, bandgap opening and pseudo magnetic field generation. However, most of these predictions have not yet been confirmed because it is experimentally difficult to control the magnitude and type (e.g., uniaxial, biaxial, and so forth) of strain in graphene devices. Here we report two novel methods to apply strain without bending the substrate. We employ thin films of evaporated metal and organic insulator deposited on graphene, which shrink after electron beam irradiation or heat application. These methods make it possible to apply both biaxial strain and in-plane isotropic compressive strain in a well-controlled manner. Raman spectroscopy measurements show a clear splitting of the degenerate states of the G-band in the case of biaxial strain, and G-band blue shift without splitting in the case of in-plane isotropic compressive strain. In the case of biaxial strain application, we find out the ratio of the strain component perpendicular to the stretching direction is at least three times larger than what was previously observed, indicating that shrinkage of the metal or organic insulator deposited on graphene induces both tensile and compressive strain in this atomically thin material. Our studies present for the first time a viable way to apply strain to graphene without the need to bend the substrate.
Project description:Tensile forces generated by stress fibers drive signal transduction events at focal adhesions. Here, we report that stress fibers per se act as a platform for tension-induced activation of biochemical signals. The MAP kinase, ERK is activated on stress fibers in a myosin II-dependent manner. In myosin II-inhibited cells, uniaxial stretching of cell adhesion substrates restores ERK activation on stress fibers. By quantifying myosin II- or mechanical stretch-mediated tensile forces in individual stress fibers, we show that ERK activation on stress fibers correlates positively with tensile forces acting on the fibers, indicating stress fibers as a tension sensor in ERK activation. Myosin II-dependent ERK activation is also observed on actomyosin bundles connecting E-cadherin clusters, thus suggesting that actomyosin bundles, in general, work as a platform for tension-dependent ERK activation.